This invention relates to an assay; more particularly, it relates to a method for the determination of fructosamines or other glycated proteins in biological materials.
Fructosamines are glycated proteins, present in biological materials, for example blood serum. "Glycation" is defined as the non-enzymatic glycosylation of proteins, such as serum albumin, by the condensation of reducing sugars, such as glucose, with the protein, (see Roth, M., (1983), Clin. Chem., 29, 1991). The reaction of glucose with albumin involves the nucleophilic attack of the carbonyl group of glucose on free amino groups on the protein. The thus-formed Schiff base may hydrolyse back to glucose and protein or it may undergo an Amadori rearrangement, (see Hodge, J.E., (1955), Adv. Carbohydr. Chem., 10, 169-205), to form a ketoamine structure. This reaction sequence is illustrated in accompanying FIG. 1. The Amadori compound is stabilised by equilibration of the linear ketoamine structure into several cyclic, hemiketal conformations in solution. The principal sites of glycation are the .epsilon.-amino groups of lysine residues and the .alpha.-amino group of the protein's terminal amino acid. Once formed, the stable ketoamine structure remains with the protein throughout its life-span.
Many disease states are characterised by unusually high or low levels of specific components of the body's metabolism. If the normal concentration range of a component in a healthy population is known then the detection of abnormal levels of this component provides a useful indication of metabolic disorder caused by disease. The purpose of clinical diagnostic tests, therefore, is to allow the performance of qualitative and quantitative analysis on body fluids, such as blood, urine and spinal fluid, as well as on tissue and other materials. The information obtained from these tests is useful to physicians in the monitoring and treatment of disease. For the information to be meaninfgul, the tests performed must be reliable and accurate. Generally, diagnostic assays make use of some unique chemical property of the analyte as the basis of the assay method. A sample of the body fluid or other material containing the analyte to be measured, generally after a suitable work-up, is contacted with a reagent which is designed to interact with the analyte in a specific way so that a measurable signal is produced. Thus, a chemical assay would involve a reagent that reacts with the analyte in a measurable way, without reacting with other components of the sample. Ideally, the reaction between the reagent and the analyte should be so specific that no other substances will react in the same manner. However, in chemical based assays, this is seldom the case and interfering side reactions are often a problem.
This problem may frequently be overcome by designing an enzyme based assay. Enzymes, by the very nature thereof, are highly specific for their substrate molecules. Although an enzyme depends on the chemical properties of its substrate to perform a specific reaction the enzyme must first recognise the physical and chemical "shape" of the substrate so that binding may occur. Only then may the enzymic reaction take place. In an enzyme based assay, therefore, a reagent containing an enzyme specific for the analyte is usually used to bind and transform the analyte in a way that is measurable. Enzyme based diagnostic assays may therefore offer advantages of specificity over chemical methods.
The level of fructosamine present in blood is governed by the concentration of sugars, such as glucose, in solution in serum. As fructosamines have a half-life of 2-3 weeks in serum, the level of fructosamine present reflects the average blood glucose levels over a period of 1-3 weeks. Thus, measurement of this parameter is a useful means of monitoring glycaemic control in diabetes mellitus.
At present, there are several established non-enzymic methods for measuring levels of serum fructosamines. For example, one method involves the separation of glycated from unglycated proteins by affinity chromatography, (see Diabetes, (1980) , 29, 1044-1047).
Immobilised m-aminophenyl-boronic acid complexes with the cis-diol groups of the glycating sugars under alkaline conditions. Unbound materials are removed by washing with buffer and the fructosamines are eluted by high concentrations of sorbitol. The levels of fructosamine in the eluent may then be measured by absorbance at 280 nm or by chemical methods. The disadvantages of such a method are that free glucose must first be removed from the samples and that the amount of glycated protein that binds to the immobilised m-aminophenyl-boronic acid is critically dependant on chromatographic conditions. This may therefore reduce the accuracy of the method.
Another known method involves the detection of the breakdown products of acid hydrolysis of the ketoamine bonds. Treatment of glycated proteins with strong acids at elevated temperatures, such as 6 mol/l HCl at 95.degree. C., causes hydrolysis of the glycated lysine residues and yields a specific product, N-(2-furoylmethyl)-L-lysine (furosine). Furosine is measured by HPLC using a reverse phase column and simultaneous UV detection at 254 and 280 nm, (see J. Clin. Chem. Clin. Biochem., (1981), 19, 81-87). Human serum albumin containing a known amount of glycated lysine residues is used for calibration. However, the method is time consuming and unsuitable for routine work or automation.
Acid hydrolysis of fructosamine is also used in another method in which treatment with weak or diluted acids yields 5-hydroxymethyl-2-furfuraldehyde. This product may be determined spectrophotometrically at 280 nm after HPLC separation. However, a more convenient method involves the reaction of the furfural product with 2-thiobarbituric acid, which results in a derivative with an absorbance maximum at 443 nm (see FEBS Lett., (1976), 71, 356-360). This procedure has been partially automated using dedicated equipment; however, the accuracy of the results depends on several factors including the level of protein in the samples, the conditions of the acid hydrolysis and the removal of glucose.
A further method which has recently replaced many of the above procedures depends on the reducing ability of fructosamine in alkaline solutions. One such method involves the addition of a serum sample to carbonate buffer, pH 10.35, containing nitroblue tetrazolium (NBT). The NBT is reduced, probably via a superoxide radical intermediate, and the absorbance of the formazan product is measured at 550 nm. The method relies on the observation that most interfering components in serum react in the first 10 minutes and hence specific serum reducing activity is measured between 10 and 15 minutes. The procedure is rapid and has been automated on a variety of analysers for clinical diagnostic use. However, the specificity of the method for glycated proteins has been questioned and it has been shown that non-specific components may lead to interference and misinterpretation of the results. In addition, the fructosamine level is influenced by the level of albumin in the sample and so the results may need to be adjusted, especially in cases of hypoalbuminaemia.
An object of the present invention is to provide a method for measuring serum fructosamine levels as an indicator of diabetic control, for example, which offers significant advantages over the existing methods. In order to do this, it was necessary to provide enzymes capable of using glycated proteins as substrates.